Decellularized extracellular matrix of conditioned body tissues and uses thereof

ABSTRACT

The present invention relates generally to decellularized extracellular matrix of conditioned body tissues. The decellularized extracellular matrix contains a biological material, preferably vascular endothelial growth factor (VEGF), produced by the conditioned body tissue that is in an amount different than the amount of the biological material that the body tissue would produce absent the conditioning. The invention also relates to methods of making and methods of using said decellularized extracellular matrix. Specifically, the invention relates to treating defective, diseased, damaged or ischemic cells, tissues or organs in a subject by administering, injecting or implanting the decellularized extracellular matrix of the invention into a subject in need thereof. The invention is further directed to a tissue regeneration scaffold for implantation into a subject inflicted with a disease or condition that requires tissue or organ repair, regeneration and/or strengthening. Additionally, the invention is directed to a medical device, preferably a stent or an artificial heart, having a surface coated or covered with the decellularized extracellular matrix of the invention or having a component comprising the decellularized extracellular matrix of the invention for implantation into a subject, preferably a human. Methods for making the tissue regeneration scaffold and methods for manufacturing a coated or covered medical device having a component comprising decellularized extracellular matrix of conditioned body tissues are also provided.

1. FIELD OF THE INVENTION

The present invention relates generally to decellularized extracellularmatrix of conditioned body tissues, as well as methods for theproduction and use thereof. In particular, the invention relates totreating defective, diseased, damaged or ischemic tissues or organs in asubject by injecting or implanting decellularized extracellular matrixof conditioned body tissue into a subject in need thereof. Moreparticularly, the invention is directed to a tissue regenerationscaffold for implantation into a subject inflicted with a disease orcondition that requires tissue or organ repair, regeneration and/orstrengthening. Further, the invention is directed to a medical device,preferably a stent or an artificial heart, having a surface coated orcovered with decellularized extracellular matrix from conditioned bodytissue and/or having a component comprising the decellularizedextracellular matrix for implantation into a subject, preferably ahuman. Methods for manufacturing a coated or covered medical device andmethods for manufacturing a medical device having a component comprisingdecellularized extracellular matrix from conditioned body tissue and/ora coated or covered surface are also provided.

2. BACKGROUND OF THE INVENTION

Despite advances in medicine and healthcare, tissue and organ failureremain a frequent and costly occurrence. Each year in the United States,40 to 90 million hospital days costing about $400 billion are attributedto the treatment of tissue and organ failure (Cohen et al., 1993, Chest103(2):656). Incidents of cellular atrophy or injury to tissue and organcaused by trauma, burns, infection, inflammation, inadequate nutrition,diminished blood supply, loss of endocrine stimulation, aging, etc., arealso prevalent. It is believed that a main pathway in the formation ofcancer is considered to be repetitive tissue injury by highly chemicallyreactive free radicals and avid oxidants. Approximately eight millionprocedures are performed each year in the United States to treatpatients suffering from tissue or organ injury or failure.

Traditionally, injured or diseased tissues or organs are treated bytransplantation or through the use of a mechanical-type substitute.However, transplantation is associated with numerous complications(e.g., graft rejection, graft-versus-host disease) while mechanicalsubstitutes only provide interim relief. Ultimately, the idealtreatments involve repairing or regenerating the tissue or organ. Theapplication of functional genomics and developmental biology hasaccelerated tissue engineering product development by elucidatingmechanisms of repair and regeneration. The use of animal products in thecreation of tissue engineering products has provided important materialsfor the treatment, management or prevention of diseases or disordersthat affect tissues and organs.

Soft tissue implantation represents an important step in tissue andorgan healing. Soft tissue implants (as opposed to orthopedic, or hardtissue, implants), include biomaterials, synthetic materials, andtissues harvested from animals. The use of soft tissue implants areespecially significant in the field of plastic and reconstructivesurgery (Tarnow et al., 1996, J. Esthet. Dent. 8(1):12-9). For example,soft tissue implants can be used to reconstruct surgically ortraumatically created tissue voids, to restore bulk to aging tissues, tocorrect soft tissue folds, and to augment tissue for cosmeticenhancement.

Diseased or damaged tendons, cartilage, and ligaments, on the otherhand, are currently treated using orthopedic or hard tissue implants.Other treatment options include stimulation of bone marrow to formrepair tissue, transplantation of osteochondral autografts orallografts, implantation of cultural autologous chondrocyctes, and useof resorbable scaffolding (with or without cells).

In response to the need for more efficient and effective implantmaterials, the use of extracellular matrix (ECM) as templates for tissueor organ repair or regeneration has increased (Schmidt and Baier, 2000,Biomaterials 21:2215-31). Although the exact mechanisms through whichECM facilitates repair or regeneration are not known, the compositionand the organization of the components are considered to be importantfactors that influence cell proliferation, gene expression patterns, andcell differentiation.

ECM is a complex structural entity surrounding and supporting cells. Theextracellular matrix is found within mammalian tissues and is made up ofthree major classes of biomolecules: structural proteins (e.g., collagenand elastin), specialized proteins (e.g., fibrillin, fibronectin, andlaminin), and proteoglycans (e.g., glycosaminoglycans). In addition toproviding physical support to cells, the extracellular matrix affectscell function through mechanical and chemical signals.

Recent findings show porcine-derived, xenogeneic extracellular matrixderived from either the small intestinal submucosa or urinary bladdersubmucosa are useful as a tissue scaffold for esophageal repair inanimal models (Badylak et al, 2000, J. Pediatr. Surg. 35(7):1097-10).Other studies have also shown that extracellular matrix derived from thesubmucosa of the porcine small intestine induces angiogenesis and hosttissue remodelling when used as a xenogeneic bioscaffold in animalmodels of wound repair (Hodde et al, 2001, Endothelium 8(1):11-24).Cytokine analysis demonstrates that xenogeneic extracellular matrixgrafts minimizes inflammatory response due to rejection (Allman et al,2001, Transplantation 71(11):1631-40).

Despite current uses of extracellular matrix for tissue or organ repairor regeneration, it is often desirable that the extracellular matrixused for treatment contain an excess amount or a specific ratio of aparticular protein, such as a growth hormone, preferably vascularendothelial growth factor (VEGF), to promote tissue growth, than thatwhich naturally occurs in the extracellular matrix. There is a continuedlack of suitable material that provides the best combination ofbiologically active materials and/or a desirable histoarchitecture as animplant to repair, regenerate or strengthen tissue or organs. There hasyet to be developed a completely biocompatible, long-lasting implantthat promotes and/or expedites tissue or organ repair or regeneration.Hence, the goal of the present invention is to provide body implantsthat are engineered for a specific application for a specific tissue ororgan (i.e., an implant that provides a specific composition ofbiologically active material and mechanical properties).

3. SUMMARY OF THE INVENTION

To achieve the aforementioned objectives, we have invented an injectableor implantable composition comprising decellularized extracellularmatrix obtained from conditioned body tissue of a donor subject. Inparticular, the invention relates to methods for producing thedecellularized extracellular matrix by conditioning body tissue from adonor animal to produce a biological material, allowing the conditionedbody tissue to produce the biological material, harvesting theconditioned body tissue from the donor animal, and decellularizing theharvested and conditioned body tissue to obtain the extracellular matrixcontaining the biological material.

In certain embodiments, the body tissue is conditioned in vivo or insitu before being harvested. In certain other embodiments, the bodytissue is conditioned in vitro after being harvested. If the body tissueis conditioned in vivo or in situ, conditioning may be performed locallyor systemically. If the body tissue is conditioned in vitro,conditioning may be performed in a bioreactor. The conditioned bodytissue is given a period of time before and/or after harvest to producethe biological material in an amount of interest. The amount ofbiological material produced by the body tissue may be monitored before,during or after the conditioning step.

The body tissue may be conditioned using any one or more biological,chemical, pharmaceutical, physiological and/or mechanical treatment(s).In one embodiment, the body tissue is biologically conditioned bytransfecting the body tissue with a nucleic acid. In another embodiment,the body tissue is chemically conditioned by incubating the body tissuein a hypotonic or hypertonic solution. In yet another embodiment, thebody tissue is pharmaceutically conditioned by delivering a therapeuticagent to the body tissue. In yet another embodiment, the body tissue isphysiologically conditioned by exposing the body tissue to heat shock.In yet another embodiment, the body tissue is mechanically conditionedby applying a force to the body tissue. Preferably, the force isproduced by the expansion of a balloon against the body tissue.

The body tissue from a donor subject may be conditioned so that thebiochemical composition and histoarchitecture of the body tissue isretained. In certain embodiments, the body tissue may be conditioned sothat the biochemical composition and histoarchitecture of the bodytissue from the donor subject is similar to the body tissue that isbeing repair, replaced and/or regenerated in a recipient subject. Thebody tissue may be from a mammal, preferably a pig or human.

The conditioned body tissue may retain or possess new physicalproperties such as strength, resiliency, density, insolubility, andpermeability as compared to the unconditioned body tissue. Theconditioned body tissue may also contain a biological material in anamount different than the amount of the biological material that thebody tissue would produce absent the conditioning. In a specificembodiment, the biological material is a growth factor, preferablyvascular endothelial growth factor (VEGF). In another specificembodiment, the biological material is an extracellular matrix protein,preferably elastin.

The harvested and conditioned body tissue may be decellularized using acombination of physical, chemical, and biological processes. The methodsof the present invention involve the steps of decellularization byremoving native cells, antigens, and cellular debris from theextracellular matrix of the body tissue. Preferably, an enzyme treatingstep is involved.

The body tissue may be further processed after decellularization tofacilitate administration, injection or implantation. For examples, thedecellularized extracellular matrix can be dried, concentrated, diluted,lyophilized, cryopreserved, electrically charged, sterilized, etc. In apreferred embodiment, the decellularized extracellular matrix issuspended in a saline solution as a final product.

The invention also relates to the administration, injection orimplantation of the decellularized extracellular matrix of conditionedbody tissue into a subject in need thereof. The decellularizedextracellular matrix of the invention may be administered, injected orimplanted alone or in combination with other therapeutically orprophylactically effective agents useful for treating, managing orpreventing a disease or condition that requires tissue or organ repair,restoration and/or strengthening may be delivered to the body tissuebefore and/or after conditioning and/or harvesting.

The decellularized extracellular matrix may also be administered,injected or implanted before, during or after treatment with othermethods of repairing, regenerating and/or strengthening of the diseased,defected, damaged or ischemic tissue or organ. In particular, thedecellularized extracellular matrix may be used to promote angiogenesisand/or repair, replace or regenerate cells, tissues or organs, such asbut not limited to lymph vessels, blood vessels, heart valves,myocardium, pericardium, pericardial sac, dura mater, meniscus, omentum,mesentery, conjunctiva, umbilical cords, bone marrow, bone pieces,ligaments, tendon, tooth implants, dermis, skin, muscle, nerves, spinalcord, pancreas, gut, intestines, peritoneum, submucosa, stomach, liver,and bladder.

The decellularized extracellular matrix of the present invention canalso be used to form a tissue regeneration scaffold for implantationinto a subject. The tissue regeneration scaffold may be used as atherapeutics to treat diseases or conditions that may benefit fromimproved angiogenesis, cell proliferation and/or tissue regenerationand/or strengthening. Such diseases or conditions include but are notlimited to, burns, ulcer, trauma, wound, bond fracture, diabetes,psoriasis, arthritis, asthma, cystitis, inflammation, infection,ischemia, restenosis, stricture, atherosclerosis, occlusion, stroke,infarct, aneurysm, abdominal aortic aneurysm, uterine fibroid, urinaryincontinence, vascular disorders, hemophilia, cancer, and organ failure(e.g., heart, kidney, lung, liver, intestine, etc.).

The invention further relates to a medical device comprisingdecellularized extracellular matrix of conditioned body tissue andmethods for manufacturing such a medical device. The medical device issuitable for insertion into a subject, preferably a human. Preferably,the medical device is non-biodegradable. More preferably, the medicaldevice is a stent or an artificial heart. In a specific embodiment, thedecellularized extracellular matrix is coated onto the medical device.Preferably, the decellularized extracellular matrix is coated onto themedical device by spray coating, with or without a polymer carrier, ordip coating. In another specific embodiment, the decellularizedextracellular matrix is used to construct a component of the medicaldevice such as a wired-like elements of a stent or a valve of anartificial heart. The decellularized extracellular matrix may be usedalone or in combination with a bulk polymer or biologically activematerial, preferably paclitaxel, to make, cover or coat the medicaldevice.

3.1 Definitions

As used herein, the term “therapeutically effective amount” refers tothat amount of the therapeutic agent sufficient to treat or managedefective, diseased, damaged or ischemic tissues or organs. Atherapeutically effective amount may refer to the amount of therapeuticagent sufficient to delay or minimize the onset of symptoms associatedwith defective, diseased, damaged or ischemic tissues or organs. Atherapeutically effective amount may also refer to the amount of thetherapeutic agent that provides a therapeutic benefit in the treatmentor management of the defective, diseased, damaged or ischemic tissues ororgans. Further, a therapeutically effective amount with respect to atherapeutic agent of the invention means that amount of therapeuticagent alone, or in combination with other agents or therapies, thatprovides a therapeutic benefit in the treatment or management ofdefective, diseased, damaged or ischemic tissues or organs. Used inconnection with an amount of the decellularized extracellular matrix ofthe invention, the term can encompass an amount that improves overalltherapy, reduces or avoids unwanted effects, or enhances the therapeuticefficacy of or synergies with another therapeutic agent.

As used herein, the term “prophylactically effective amount” refers tothat amount of the prophylactic agent sufficient to result in theprevention of the occurrence of defective, diseased, damaged or ischemictissues or organs. A prophylactically effective amount may also refer tothe amount of prophylactic agent sufficient to prevent the occurrence orrecurrence of defective, diseased, damaged or ischemic tissues or organsin a patient, including but not limited to those (genetically)predisposed. A prophylactically effective amount may also refer to theamount of the prophylactic agent that provides a prophylactic benefit inthe prevention of defective, diseased, damaged or ischemic tissues ororgans. Further, a prophylactically effective amount with respect to aprophylactic agent of the invention means that amount of prophylacticagent alone, or in combination with other agents or therapies, thatprovides a prophylactic benefit in the prevention of the occurrence orrecurrence of defective, diseased, damaged or ischemic tissues ororgans. Used in connection with an amount of the decellularizedextracellular matrix of the invention, the term can encompass an amountthat improves overall prophylaxis or enhances the prophylactic efficacyof or synergies with another prophylactic agent.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) anda primate (e.g., monkey and human), most preferably a human.

As used herein, the term “body tissue” broadly encompasses any or anumber of cells, tissues or organs.

As used herein, the term “repair” relates to the restoration ofdefective, diseased, damaged or ischemic tissues or organs to a sound orhealthy stage by replacing a part or putting together what is defective,diseased, damaged or ischemic by synthesizing and incorporatingadditional normal cells, tissue or organ components to increase the sizeand/or strength of the defective, diseased, damaged or ischemic tissueor organ.

As used herein, the term “replace” relates to the substitution ofdefective, diseased, damaged or ischemic tissues or organs with newlysynthesized cells, tissue or organ components facilitated by thedecellularized extracellular matrix of the present invention.

As used herein, the term “regenerate” relates to the regrowth and/orreconstitution of defective, diseased, damaged or ischemic tissues ororgans.

As used herein, the term “strengthen” relates to the making stronger ofthe defective, diseased, damaged or ischemic tissues or organs.

As used herein, the terms “biological material” and “biologically activematerial” are used interchangeably. Examples of a biological materialinclude, but are not limited to, vascular endothelial growth factor(VEGF), transforming growth factor (TGF), fibroblast growth factor(FGF), epidermal growth factor (EGF), cartilage growth factor (CGF),nerve growth factor (NGF), keratinocyte growth factor (KGF), skeletalgrowth factor (SGF), osteoblast-derived growth factor (BDGF), hepatocytegrowth factor (HGF), insulin-like growth factor (IGF), cytokine growthfactors (CGF), platelet-derived growth factor (PDGF), hypoxia induciblefactor-1 (HIF-1), stem cell derived factor (SDF), stem cell factor(SCF), endothelial cell growth supplement (ECGS), granulocyte macrophagecolony stimulating factor (GM-CSF), growth differentiation factor (GDF),integrin modulating factor (IMF), calmodulin (CaM), thymidine kinase(TK), tumor necrosis factor (TNF), growth hormone (GH), bone morphogenicproteins (BMP), matrix metalloproteinase (MMP), tissue inhibitor ofmatrix metalloproteinase (TIMP), cytokines, interleukins, lymphokines,interferon, integrin, collagen (all types), elastin, fibrillins,fibronectin, laminin, glycosaminoglycans, vitronectin, proteoglycans,transferrin, cytotactin, cell binding domains (e.g., RGD), and tenascin.

As used herein, the term “analog” refers to a polypeptide that possessesa similar or identical function as a particular protein (e.g., vascularendothelial growth factor), or a fragment thereof, but does notnecessarily comprise a similar or identical amino acid sequence orstructure of that protein or a fragment thereof. A polypeptide that hasa similar amino acid sequence refers to a polypeptide that satisfies atleast one of the following: (a) a polypeptide having an amino acidsequence that is at least 30%, at least 35%, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the amino acid sequence of a protein or afragment thereof as described herein; (b) a polypeptide encoded by anucleotide sequence that hybridizes under stringent conditions to anucleotide sequence encoding a protein or a fragment thereof asdescribed herein of at least 20 amino acid residues, at least 30 aminoacid residues, at least 40 amino acid residues, at least 50 amino acidresidues, at least 60 amino residues, at least 70 amino acid residues,at least 80 amino acid residues, at least 90 amino acid residues, atleast 100 amino acid residues, at least 125 amino acid residues, or atleast 150 amino acid residues; and (c) a polypeptide encoded by anucleotide sequence that is at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or at least 99% identical to the nucleotide sequence encodinga protein or a fragment thereof as described herein. A polypeptide withsimilar structure to a protein or a fragment thereof as described hereinrefers to a polypeptide that has a similar secondary, tertiary orquaternary structure of a protein or a fragment thereof as describedherein. The structure of a polypeptide can be determined by methodsknown to those skilled in the art, including but not limited to, X-raycrystallography, nuclear magnetic resonance, and crystallographicelectron microscopy.

As used herein, the term “derivative” refers to a polypeptide thatcomprises an amino acid sequence of a protein, such as vascularendothelial growth factor, a fragment of the protein, an antibody thatimmunospecifically binds to the protein, or an antibody fragment thatimmunospecifically binds to the protein which has been altered by theintroduction of amino acid residue substitutions, deletions oradditions. The term “derivative” as used herein also refers to theprotein, a fragment of the protein, an antibody that immunospecificallybinds to the protein, or an antibody fragment that immunospecificallybinds to the protein which has been modified, i.e., by the covalentattachment of any type of molecule to the polypeptide. For example, butnot by way of limitation, by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. A derivative may also be modified by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Further, thederivative may contain one or more non-classical amino acids. In oneembodiment, the derivative possesses a similar or identical function asthe protein of interest. In another embodiment, the derivative has analtered activity when compared to an unaltered protein. For example, aderivative antibody or fragment thereof can bind to its epitope moretightly or be more resistant to proteolysis.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 20 contiguous amino acidresidues, at least 30 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of aprotein, such as vascular endothelial growth factor.

The percent identity of two amino acid sequences or of two nucleic acidsequences is determined by aligning the sequences for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence for bestalignment with the sequence) and comparing the amino acid residues ornucleotides at corresponding positions. The “best alignment” is analignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity=# of identical positions/total # of positions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul et al., 1990, J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Myers and Miller, CABIOS (1989). TheALIGN program (version 2.0) which is part of the CGC sequence alignmentsoftware package has incorporated such an algorithm. Other algorithmsfor sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5;and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to decellularized extracellular matrix ofconditioned body tissue. In certain embodiments, the body tissue of adonor subject is conditioned in vivo or in situ before harvest. Incertain embodiments, the body tissue of a donor subject is firstharvested and then conditioned in vitro, preferably in a bioreactor. Theconditioned body tissue is given a period of time to produce abiological material in an amount different than the amount that isproduced by a body tissue absent the conditioning. The conditioned bodytissue may be decellularized by at least one or a combination ofphysical, chemical and/or biological step(s). Preferably, thedecellularized conditioned body tissue is rid of cellular components andonly retains the extracellular matrix and the biological material ofinterest. In certain embodiments, the decellularized conditioned bodytissue can be further processed prior to its use.

The decellularized extracellular matrix may be grafted directly onto thesite of a defective, diseased, damaged or ischemic tissue or organ. Thedecellularized extracellular matrix may also be processed into aformulation and injected at a site in need of treatment. Thedecellularized extracellular matrix may further be used in a tissueregeneration scaffold for implantation into a subject. In addition, thedecellularized extracellular matrix can be part of a medical device,preferably a stent or an artificial heart, for implantation into asubject. For instance, the decellularized extracellular matrix can becoated onto the medical device, preferably by spray coating or dipcoating, or incorporated into a component of the medical device.

Although not to be limited in theory, the decellularized extracellularmatrix provides a microenvironment and contains important biologicalmaterials that promote the efficient and effective repair, regenerationand/or strengthening of cells, tissues or organs.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

4.1 Decellularized Extracellular Matrix of Conditioned Body Tissue

4.1.1 Source of Body Tissue

Suitable animal body tissue from which the decellularized extracellularmatrix material of the present invention is produced includes bodytissues originally from syngeneic, allogeneic or xenogenic sources. Thebody tissue may be obtained from various animal sources. These animalsinclude, but are not limited to, non-primate (e.g., cows, pigs, horses,chickens, cats, dogs, rats, etc.) and primate (e.g., monkeys andhumans). The body tissue may be obtained at approved slaughterhousesfrom animals fit for human consumption or from herds of domesticatedanimals maintained for the purpose of providing tissues or organs.Preferably, the body tissue is handled in a sterile manner, and anyfurther dissection of the body tissue is carried out under asepticconditions. A preferred source of the body tissue is human. When theimplants are obtained from human, the donor may be the recipient, or thedonor may be genetically related to the recipient. In specificembodiments, the donor is tested for competency with the recipient.

Progenitor cells (e.g., endothelial progenitor cells), stem cells (e.g.,mesenchymal, hematopoietic, neuronal), stromal cells, parenchymal cells,undifferentiated cells, embryonic cells, fibroblasts, macrophage, andsatellite cells are particularly preferred for conditioning using themethods of the present invention. In preferred embodiments, body organsthat are useful in the present invention include, but are not limitedto, brain, heart, lung, liver, pancreas, stomach, large or smallintestine, kidney, bladder, uterus, bone marrow, etc.

The body tissue suitable for the present invention can be grouped intofour general categories: (1) epithelial tissue, (2) connective tissue,(3) muscle tissue, and (4) nerve tissue. Epithelial tissue covers orlines all body surfaces inside or outside the body. Examples ofepithelial tissue include, but are not limited to, the skin, epithelium,dermis, and the mucosa and serosa that line the body cavity and internalorgans, such as the heart, lung, liver, kidney, intestines, bladder,uterine, etc. Connective tissue is the most abundant and widelydistributed of all tissues. Examples of connective tissue include, butare not limited to, vascular tissue (e.g., arteries, veins,capillaries), blood (e.g., red blood cells, platelets, white bloodcells), lymph, fat, fibers, cartilage, ligaments, tendon, bone, teeth,omentum, peritoneum, mesentery, meniscus, conjunctiva, dura mater,umbilical cord, etc. Muscle tissue accounts for nearly one-third of thetotal body weight and consists of three distinct subtypes: striated(skeletal) muscle, smooth (visceral) muscle, and cardiac muscle.Examples of muscle tissue include, but are not limited to, myocardium(heart muscle), skeletal, intestinal wall, etc. The fourth primary typeof tissue is nerve tissue. Nerve tissue is found in the brain, spinalcord, and accompanying nerve. Nerve tissue is composed of specializedcells called neurons (nerve cells) and neuroglial or glial cells.

4.1.2 Conditioning of Body Tissue

The present invention provides methods for conditioning body tissueusing one or more biological, chemical, pharmaceutical, physiologicaland/or mechanical manipulation. Specifically, conditioning is used tomake the body tissue either over-express or under-express a biologicalmaterial of interest as compared to the amount of such biologicalmaterial that the body tissue would express absent conditioning, or toexpress a protein or biological material otherwise not present in thetissue. In certain embodiments, the conditioning modify the productionof biological materials that enhance the effectiveness or temporalsequence of repairing, regenerating or strengthening defective,diseased, damaged or ischemic tissues or organs in a subject. In certainother embodiments, the conditioning modify the production of biologicalmaterials that increase the metabolic synthesis of and/or phenotypicexpression in endogenous cell populations. The anti-adhesion,bioadhesive, bioresorptive, antithrombogenic, and other physicalproperties of the body tissue can also be varied as needed by theconditioning process.

Preferably, the conditioning modifies the body tissue's production ofextracellular matrix proteins, growth factors, angiogenesis factors,cytokines, morphogens (a biologically active material that is capable ofinducing the developmental cascade of cellular and molecular events thatculminate in the formation of new, organ-specific tissue), etc., and/ormicro-architecture of extracellular matrix components. Examples of thebiological material of interest to the present invention include, butare not limited to, vascular endothelial growth factor (VEGF),transforming growth factor (TGF), fibroblast growth factor (FGF),epidermal growth factor (EGF), cartilage growth factor (CGF), nervegrowth factor (NGF), keratinocyte growth factor (KGF), skeletal growthfactor (SGF), osteoblast-derived growth factor (BDGF), hepatocyte growthfactor (HGF), insulin-like growth factor (IGF), cytokine growth factors(CGF), platelet-derived growth factor (PDGF), hypoxia inducible factor-I(HIF-1), stem cell derived factor (SDF), stem cell factor (SCF),endothelial cell growth supplement (ECGS), granulocyte macrophage colonystimulating factor (GM-CSF), growth differentiation factor (GDF),integrin modulating factor (IMF), calmodulin (CaM), thymidine kinase(TK), tumor necrosis factor (TNF), growth hormone (GH), bone morphogenicprotein (BMP) (e.g., BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7(PO-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-14, BMP-15, BMP-16,etc.), matrix metalloproteinase (MMP), tissue inhibitor of matrixmetalloproteinase (TIMP), cytokines, interleukin (e.g., IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15,etc.), lymphokines, interferon, integrin, collagen (all types), elastin,fibrillins, fibronectin, vitronectin, laminin, glycosaminoglycans,proteoglycans, transferrin, cytotactin, cell binding domains (e.g.,RGD), and tenascin.

More than one conditioning process may be performed, sequentially orsimultaneously. The conditioning of the body tissue can be conducted invivo, in situ or in vitro. Conditioning the body tissue while it isstill in the donor animal has the advantage of retaining the complexityafforded by in vivo remodelling.

Alternatively, after the body tissue is harvested, the biologicallyactive material composition and histoarchitectural property of the bodytissue may be modified without in vivo manipulation. When conditioningis performed after the body tissue is isolated or harvested from thedonor animal, i.e., in vitro, the body tissue is cultured for a periodof time, for example, in a bioreactor. The advantage of in vitroconditioning is that the process is easily monitored and that changes tothe biologically active material composition and histoarchitecturalproperty of the body tissue is easily assessed.

Regardless of whether the body tissue is conditioned in vivo or invitro, or before or after the body tissue is harvested, the conditionedbody tissue should be allowed a selected period of time to produce thedesired biological material in an amount different than the amount thatis produced by an unconditioned body tissue. Preferably, the conditionedbody tissue produces at least 5%, at least 10%, at least 25%, at least50%, at least 100%, at least two times, at least five times, or at leastten times more or less biological material than a body tissue absentconditioning.

In another embodiment, the body tissue is conditioned to express aprotein or biological material otherwise not present in the tissue.

In certain preferred embodiments, the conditioned body tissue can befurther processed before or after decellularization. In a specificembodiments, a therapeutic agent may be delivered to the body tissuebefore or after conditioning. Preferably, the therapeutic agent isuseful for treating a disease or condition that requires tissue or organrepair, restoration and/or strengthening.

4.1.2.1 Biological Conditioning

The body tissue of a donor animal can be biologically conditioned bygenetic engineering to effect a desired change in composition or amountof biologically active material in the body tissue. For instance, thebody tissue may be transfected with a nucleic acid that encodes abiological material of interest (see International Publication No. WO98/28406). The body tissue of a donor animal can also be biologicallyconditioned using a number of in vitro culture conditions to effectchanges to the histoarchitecture of the body tissue and/or compositionof biologically active materials in the body tissue. In preferredembodiments, the in vitro biological conditioning includes the use of abioreactor. In a specific embodiment, the conditioned body tissue iscontinuously cultured in the bioreactor while toxic metabolic byproductsare removed.

In general, cells in the body tissue of an animal can be transfected invivo or in vitro with genetic material using any appropriate means suchas direct injection of viral vectors, as discussed further in detailbelow, delivery into the local blood supply (see InternationalPublication Nos. WO 98/58542 and WO 99/55379, each of which isincorporated herein by reference in its entirety), the use of deliveryvectors (e.g., liposome) or chemical transfectants, andphysico-mechanical methods such as electroporation and direct diffusionof nucleic acid. The transfected body tissue is subsequently culturedfor a period of time during which the composition or amount of at leastone biological material in the body tissue is changed.

For general reviews of the methods of gene transfer, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217, each of which is incorporated herein byreference in its entirety. Delivery of the nucleic acid into a donorbody tissue may be either in vivo, in which case the donor body tissueis exposed to the nucleic acid or nucleic acid-carrying vector ordelivery complex before being harvested from the donor animal; or invitro, in which case, the donor body tissue may first be harvested fromthe donor animal and then transformed with the nucleic acid in vitro.These two approaches are known, respectively, as in vivo or in vitrogene transfer.

In one embodiment, the nucleic acid is directly administered in vivo,where it is expressed to produce a biologically active material. Thiscan be accomplished by any of numerous methods known in the art, e.g.,by constructing it as part of an appropriate nucleic acid expressionvector and administering it so that it becomes intracellular, byinfection using a defective or attenuated retroviral or other viralvector (see infra. and U.S. Pat. No. 4,980,286), by direct injection ofnaked DNA, by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), by encapsulation in biopolymers(poly-β-1-4-N-acetylglucosamine polysaccharide; see U.S. Pat. No.5,635,493), by administering it in linkage to a peptide or ligand whichis known to enter the nucleus, by receptor-mediated endocytosis (see,e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), or by coating withlipids.

Viral vectors include adenoviruses, gutted adenoviruses,adeno-associated virus, retroviruses, alpha virus (Semliki Forest,Sindbis, etc.), lentiviruses, herpes simplex virus, replicationcompetent viruses (e.g., ONYX-015), and hybrid vectors. Non-viralvectors include artificial chromosomes and mini-chromosomes, plasmid DNAvectors (e.g., pCOR), cationic polymers (e.g., polyethyleneimine,polyethyleneimine (PEI)) graft copolymers (e.g., polyether-PEI andpolyethylene oxide-PEI), neutral polymers PVP, SP1017 (SUPRATEK), lipidsor lipoplexes, nanoparticles and microparticles with and withouttargeting sequences such as the protein transduction domain (PTD).

Adenoviruses, in particular, are especially attractive vehicles fordelivering genes to respiratory epithelia where they cause a milddisease. Other targets for adenovirus-based delivery systems are liver,the central nervous system, endothelial cells, and muscle. The use ofadenoviruses has the advantage of being capable of infectingnon-dividing cells. Kozarsky and Wilson present a review ofadenovirus-based gene transfer (1993, Current Opinion in Genetics andDevelopment 3:499-503). Bout et al. demonstrate the use of adenovirusvectors to transfer genes to the respiratory epithelia of rhesus monkeys(1994, Human Gene Therapy 5:3-10). Other instances of the use ofadenoviruses in gene transfer can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234. Adeno-associatedvirus (AAV) has also been proposed for use in gene transfer (see Walshet al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).

Genetically ex vivo modified cells (e.g., stem cells, fibroblasts,myoblasts, satellite cells, pericytes, cardiomyocytes, skeletalmyocytes, macrophage) may be delivered to the tissue. The cells thencondition the matrix.

Another way to transport the gene that encodes the biologically activematerial into the body tissue involves chemical or physical treatment ofthe cells in the body tissue to increase the potential for gene uptakeand allowing the gene to be directly introduced into the nucleus ortarget the gene to a cell receptor. In certain embodiments, theseinclude the use of vectors that exploit receptors on the surface ofcells using liposomes, lipids, ligands for specific surface receptors,cell receptors, calcium phosphate and other chemical mediators,microinjections, electroporation, sperms, and homologous recombination.Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTION® and LIPOFECTACE®, which are formed of cationic lipids suchas N-[1-(2,3 dioleyloxy)-propyl]-nmnm-trimethylammonium chloride (DOTMA)and dimethyl dioctadecylammonium bromide (DDAB). Numerous methods formaking liposomes are also known to those skilled in the art.

In another embodiment, a nucleic acid-ligand complex can be formed inwhich the ligand comprises a fusogenic viral peptide to disruptendosomes, allowing the nucleic acid to avoid lysosomal degradation. Inyet another embodiment, the nucleic acid can be targeted for cellspecific uptake and expression, by targeting a specific receptor (see,e.g., International Publications Nos. WO 92/06180, WO 92/22635,WO92/20316, and WO93/14188, each of which is incorporated herein byreference in its entirety). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

The invention also relates to a method for biologically conditioningbody tissue by inoculating the body tissue with a solution havingmicroorganisms, where the microorganisms are selected to producechemicals that process the tissue. The body tissue is incubated with theinoculated microorganisms under conditions that are effective forprocessing the body tissue by the chemicals produced by themicroorganisms. The body tissue may be subsequently treated tosubstantially remove or inactivate the microorganisms (see U.S. Pat. No.6,121,041).

In other embodiments, the tissue or organ may be transformed with one ormore different recombinant nucleic acid molecules, so that the cellswithin the tissue or organ may express at least one recombinant protein.In another embodiment, a single cell in the tissue or organ may betransfected with a single recombinant nucleic acid molecule thatexpresses at least one protein, which can be under the control of thesame transcription control sequences or under the control of differenttranscription control sequences. Methods commonly known in the art ofrecombinant DNA technology which may be used are described in Ausubel etal. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley &Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.(eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.

In preferred embodiments, the invention creates in the tissue or organlocalized depots for a biologically active material. The tissue or organserves to concentrate the binding of biologically active material suchas drugs that are introduced, for example, locally or systemically. Thisis accomplished by upregulating the production of anionic/cationicspecies, specific antibody recognition sequences, cell receptors, etc.,in the tissue or organ. For example, the conditioned body tissue whichcomprises cells with a highly positively charged matrix would enhancethe localization of nucleic acid at this site. This would sustainnucleic acid delivery, improve transfection and reduce degradation ofthe nucleic acid. In a specific embodiment, the depot providelocalization for biologically active material for the treatment ofischemia. In another specific embodiment, the depot provide localizationfor biologically active material listed supra and α-adrenergic blockers,β-adrenergic blockers, α-adrenergic agonists, α-1 adrenergicantagonists, AMP kinase activators, angiotensin converting enzyme (ACE)inhibitors, angiotensin II receptor antagonists, antiarrhythmic agents,anticoagulation agents, antiplatelet aggregation agents, antidiabeticagents, antioxidants, anti-inflammatory agents, beta blockers, bile acidsequestrants, calcium channel blockers, calcium antagonists, CETPinhibitors, cholesterol reducing agents/lipid regulators, drugs thatblock arachidonic acid conversion, duretics, estrogen replacementagents, inotrophic agents, fatty acid analogs, fatty acid synthesisinhibitors, fibrates, histidine, nicotine acid derivatives, nitrates,peroxisome proliferator activator receptor agonists or antagonists,ranolzine, statins, thalidomide, thiazolidinediones, thrombolyticagents, vasodilators, and vassopressors.

The form and amount of nucleic acid envisioned for use depends on thetype of biologically active material and the desired effect and can bereadily determined by one skilled in the art. For transfection of cellswithout or minimized toxic effects see U.S. Pat. No. 6,284,880.

Nucleic acids that are useful as biologically active materials for genetransfer in the present invention include, e.g., DNA and RNA sequences,that have a therapeutic or prophylactic effect after being taken up bythe cells of a tissue or an organ. In one embodiment, the nucleic acidcomprises an expression vector that expresses a biologically activematerial. In another embodiment, the nucleic acid comprises a part of anexpression vector that expresses a protein or a functionally activefragment, derivative or analog thereof, or a chimeric protein (seeInternational Publication No. WO 01/90158).

In specific embodiments, the nucleic acid encodes a sequence without aleader sequence which produces an intracellular protein. In otherspecific embodiments, the nucleic acid encodes a sequence with a leadersequence which produces an intercellular protein. In a specificembodiment, the nucleic acid encodes a biologically active material or afunctionally active fragment, derivative or analog thereof.

Preferably, the nucleic acid useful in the invention encodes forpolypeptides. A polypeptide is understood to be any translation productof a polynucleotide regardless of size, and whether modified or not. Thepolypeptide may be modified by, e.g., glycosylation, acetylation,formylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. In specific embodiments, one ormore amino acid residues in the amino acid sequence of the polypeptide,preferably non-conserved amino acid residues, may include insertion,deletion and/or substitution with a different amino acid residue. Thesepolypeptides may include, for example, those polypeptides that arebiologically active in the body tissue of the donor and/or recipientanimal.

The polypeptides, proteins, or functionally active fragments,derivatives, and analogs thereof, that are encoded by nucleic acids usedin gene transfer include without limitation, structural proteins, growthfactors and cytokines which promotes or enhances repair, regeneration orstrengthening of defective, diseased, damaged or ischemic cells, tissuesor organs.

Most preferably, genes that are useful for the present invention encodeproteins such as vascular endothelial growth factor (VEGF), transforminggrowth factor (TGF), fibroblast growth factor (FGF), epidermal growthfactor (EGF), cartilage growth factor (CGF), nerve growth factor (NGF),keratinocyte growth factor (KGF), skeletal growth factor (SGF),osteoblast-derived growth factor (BDGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), cytokine growth factors (CGF),platelet-derived growth factor (PDGF), hypoxia inducible factor-1(HIF-1), stem cell derived factor (SDF), stem cell factor (SCF),endothelial cell growth supplement (ECGS), granulocyte macrophage colonystimulating factor (GM-CSF), growth differentiation factor (GDF),integrin modulating factor (IMF), calmodulin (CaM), thymidine kinase(TK), tumor necrosis factor (TNF), growth hormone (GH), bone morphogenicproteins (BMP), matrix metalloproteinase (MMP), tissue inhibitor ofmatrix metalloproteinase (TIMP), cytokines, interleukins, lymphokines,interferon, integrin, collagen (all types), elastin, fibrillins,fibronectin, laminin, glycosaminoglycans, vitronectin, proteoglycans,transferrin, cytotactin, cell binding domains (e.g., RGD), and tenascin.Other genes that are useful in the present invention include those thatpromote angiogenesis, modulate inflammation, and increase cell adhesion,proliferation and regeneration.

In a particularly preferred embodiment, genes encoding for elastin maybe used to increase elastic properties of the tissue being implanted.The amount of elastin would be tailored to ultimately result in asuitable material for stent coatings, i e., to produce an elongationproperty necessary to comply with stent expansion.

Antisense and ribozyme molecules which inhibit expression of a targetgene can also be used in accordance with the invention. For example, ina preferred embodiment, antisense RNA molecules which inhibit theexpression of major histocompatibility gene complexes (HLA) have beenshown to be most versatile with respect to modulating immune responses.Furthermore, appropriate ribozyme molecules can be designed asdescribed, e.g., Hascloff et al., 1988, Nature 334:585-591; Zaug et al.,1984, Science 224:674-578; and Zaug and Cech, 1986, Science 231:470-475.Still further, triple helix molecules can be utilized in reducing thelevel of target gene activity. These techniques are described in detailby L. G. Davis et al., eds, Basic Methods in Molecular Biology, 2^(nd)ed., Appleton & Lange, Norwalk, Conn. 1994. Using any of the foregoingtechniques, the expression of MHC class II molecules can be knocked outin order to reduce the risk of rejection of the tissue constructsdescribed herein.

4.1.2.2 Chemical Conditioning

The body tissues may be chemically conditioned to effect a desiredchange in the composition of biologically active material and/or thehistoarchitechture of the body tissue. In one embodiment, the bodytissue may be chemically conditioned by incubating the body tissue invitro with a isosmotic, hypotonic and/or hypertonic solution (see, e.g.,U.S. Pat. No. 5,855,620 and International Publication No. WO 96/32905).Studies have shown that changes in cellular osmolality appear todirectly influence cell metabolism such as lipolysis (Bilz et al., 1999,Metabolism 48(4):472-6) or protein synthesis (Schmid, 1986, KlinWochenschr 64(1):23-8; Yates et al., 1982, J. Biol. Chem.257(24):15030-4).

In other embodiments, the body tissue may be detoxified with reducingagents including, for example, inorganic sulfur-oxygen ions, such asbisulfate and thiosulfate, organic sulfates, amines, ammonia/ammonium,and surfactants. Chemical solutions may also be added to modulate thesalinitiy, pH (acidity and alkalinity), ion concentration (e.g.,potassium, calcium, magnesium, phosphorous, sodium, nitrate, etc.),blood variables, plasma volume, and oxygen level of the body tissue tofacilitate a change in the composition or amount of biologically activematerials. Preferably, the body tissue is chemically conditioned topromote protein synthesis, cell proliferation, tissue regeneration andstrengthening or make the cells more susceptible to biological,physiological and/or mechanical conditioning.

4.1.2.3 Pharmaceutical Conditioning

Another aspect of the invention relates to the pharmaceuticalconditioning of the body tissue by delivering a therapeutic agent to thebody tissue. In one embodiment, the therapeutic agent is delivered tothe body tissue before the body tissue is harvested. In anotherembodiment, the therapeutic agent is delivered to the body tissue afterthe body tissue is harvested.

Therapeutic agents include those that are effective at treating,managing or preventing a disease or condition that requires tissue ororgan repair, restoration and/or strengthening. Other therapeutic agentsinclude those that that promote angiogenesis, modulate inflammation, andincrease cell adhesion, proliferation and regeneration. Examples oftherapeutic agents include, but are not limited to, vascular endothelialgrowth factor (VEGF), transforming growth factor (TGF), fibroblastgrowth factor (FGF), epidermal growth factor (EGF), cartilage growthfactor (CGF), nerve growth factor (NGF), keratinocyte growth factor(KGF), skeletal growth factor (SGF), osteoblast-derived growth factor(BDGF), hepatocyte growth factor (HGF), insulin-like growth factor(IGF), cytokine growth factors (CGF), platelet-derived growth factor(PDGF), hypoxia inducible factor-1 (HIF-1), stem cell derived factor(SDF), stem cell factor (SCF), endothelial cell growth supplement(ECGS), granulocyte macrophage colony stimulating factor (GM-CSF),growth differentiation factor (GDF), integrin modulating factor (IMF),calmodulin (CaM), thymidine kinase (TK), tumor necrosis factor (TNF),growth hormone (GH), bone morphogenic proteins (BMP), matrixmetalloproteinase (MMP), tissue inhibitor of matrix metalloproteinase(TIMP), cytokines, interleukins, lymphokines, interferon, integrin,collagen (all types), elastin, fibrillins, fibronectin, laminin,glycosaminoglycans, vitronectin, proteoglycans, transferrin, cytotactin,cell binding domains (e.g., RGD), tenascin, anti-inflammatory drugs,α-adrenergic blockers, β-adrenergic blockers, α-adrenergic agonists, α-1adrenergic antagonists, AMP kinase activators, angiotensin convertingenzyme (ACE) inhibitors, angiotensin II receptor antagonists,antiarrhythmic agents, anticoagulation agents, antiplatelet aggregationagents, antidiabetic agents, antioxidants, anti-inflammatory agents,beta blockers, bile acid sequestrants, calcium channel blockers, calciumantagonists, CETP inhibitors, cholesterol reducing agents/lipidregulators, drugs that block arachidonic acid conversion, duretics,estrogen replacement agents, inotrophic agents, fatty acid analogs,fatty acid synthesis inhibitors, fibrates, histidine, nicotine acidderivatives, nitrates, peroxisome proliferator activator receptoragonists or antagonists, ranolzine, statins, thalidomide,thiazolidinediones, thrombolytic agents, vasodilators, vassopressors,vitamins, antioxidants, herbal extracts, metals, etc.

The body tissue may be conditioned pharmaceutically either when thedonor subject is undergoing or has already undergone a medication ortreatment, wherein as a result of the medication or treatment, theproduction of biological materials in the body tissue is effected.

4.1.2.4 Physiological Conditioning

The body tissue may be physiologically conditioned to effect a processor function of the body tissue. In particular, the body tissue may bephysiologically conditioned to increase or decrease the level and/orrate of production of a biologically active material in the body tissueby subjecting the body tissue to temperature changes that affectchemical and protein synthesis in the cells (see e.g., Tibbett et al.,2002, Mycorrhiza 12(5):249-55).

In one embodiment, the body tissue is physiologically conditioned bycryopreservation and subsequent thawing of the body tissue as describedin U.S. Pat. No. 6,291,240, which is incorporated by reference herein inits entirety. Specifically, cryopreservation and subsequent thawing(“cryopreservation/thaw cycle”) induced the cells of the body tissue toproduce useful regulatory proteins, such as, growth factors, cytokines,and stress proteins (e.g., GRP78 and HSP90). Stress proteins are knownto stabilize cellular structures and render the cells resistant toadverse conditions.

In a specific embodiment, the tissue and organs may be cryopreserved orfrozen to below −150° C. to −180° C., preferably, to below −50° C., morepreferably, to below −65° C. to −70°C. In another specific embodiment,the body tissue may be cryopreserved by adding glycosaminoglycan orother extracellular matrix proteins and using freezing schedule designedto maximize retention of tissue cell viability and biomechanicalproperties during and after the freezing process, and following athawing schedule which maximizes cell viability. Cryopreserving agentcomprises a cell-penetrating organic solute, which is preferablydimethylsulfoxide, and a clycosaminoglycan, which is preferablychondroitin sulphate, in an amount sufficient to cryopreserve themusculoskeletal tissue such as ligaments, tendons and cartilage (seeInternational Publication No. WO 91/06213).

In yet another embodiment, the body tissue is subjected to physiologicalstresses such as oxygen deprivation or nutrient deficiency. The stressimposed on the tissue or organ by the oxygen or nutrient deprivationinduces the production of regulatory proteins in the tissue or organ andin turn changes the compositions of the biologically active material andphysical structure of the body implant.

Alternatively, U.S. Pat. No. 5,824,080 describes the use of photodynamictherapy (PDT), a technique to produce cytotoxic free radicals, was usedto condition arterial tissues. The collagens in the matrix may becross-linked using photooxidative catalysis and visible light andtherefore, add mechanical strength and/or resilience to the body tissue.

4.1.2.5 Mechanical Conditioning

Tissues responds to mechanical forces by remodelling the extracellularmatrix. The magnitude and direction of mechanical force will determinethe extent and type of remodelling. For example, increased stress onbones results in an increase in bone mass. Accordingly, artificialstressing of a tissue or organ that is to be harvested for the presentinvention modifies the properties and compositions of biologicallyactive materials of the tissue or organ. The mechanical force can berepeatedly applied over a period of time until the desired amount ofbiological active material is obtained.

In a specific embodiment, a portion of the small intestine of a donoranimal, preferably a pig, may be mechanically conditioned by placing aballoon inside the portion of the small intestine. The balloon isinflated such that it stretches the intestinal wall. Preferably, theinflation or deflation of the balloon may occur in a cyclic fashion.More preferably, the inflation only occurs during certain periods oftime during the day, thus allowing the animal's digestive system tofunction normally when the balloon is deflated.

Other methods of mechanically conditioning the body tissue includes theuse of standard clips to create tension in the body tissue. In anotherspecific embodiment, the body tissue is mechanically conditioned by theapplication of strain, wherein cell division is facilitated and theactivity of matrix metalloproteinases (MMPs) are improved (seeInternational Publication No. WO 02/62971). In yet another specificembodiment, the body tissue is subject to a hydrostatic and/orhydrodynamic force as described in U.S. Pat. No. 6,197,296, which isincorporated herein by reference in its entirety.

In yet another specific embodiment, the body tissue is subject toelectroprocessing techniques, including electrospin, electrospray,electroaerosol, and electrosputter (see International Publication Nos.WO 02/40242 and WO 02/18441). Centrifugation, electrical stimulation,electromagnetic forces (e.g., seeding tissue and/or cells with magneticparticles), hydrostatic or hydrodynamic forces, sound waves, andultrasound waves may also be used to manipulate the amount orcomposition of biologically active materials in the body tissue. In aspecific embodiment, the electrical stimulation is generated withconductive wires connected to an electric potential which cause changesby varying the electric field or by causing mechanical forces (e.g.,muscle contraction). In another specific embodiment, the electromagneticforces and/or strains are generated by applying an electromagneticfield. In another specific embodiment, the hydrostatic or hydrodynamicforces are generated by first inserting a catheter or cannula into thetissue or organ; then forcing saline or another biologically inert fluidinto the tissue and subsequently removing the same from the tissue suchthat the forces from the pressurized fluid conditions the tissue. In yetanother specific embodiment, the sound wave and ultrasound waves areproduced by commercially available spealers or transducers.

This invention also provides an in vitro method for mechanicallyconditioning tissue in an oriented manner (see U.S. Pat. Nos. 5,765,350,5,700,688 and 5,521,087). For example, connective body tissues may bealigned along a defined axis to produce an oriented tissue-equivalenthaving increased mechanical strength in the direction of the axis. Thetensile strength of collagen in a body tissue can also be improved bycross-linking or plasticizing collagen thread or thread construct with aplasticizing agent, imparting a tensile load to the collagen thread orconstruct to strain the collagen thread, and then allowing the strain inthe thread to decrease by stress-relaxation or by creep (see U.S. Pat.No. 5,718,012 and International Publication No. WO 97/45071). The amountof biological material may be measured before, during and/or after theconditioning.

Biological, chemical, or pharmaceutical conditioning may be enhanced byuse of ultrasound or iontophoresis during delivery to the tissue to beconditioned.

4.1.3 Culturing the Conditioned Body Tissue

The conditioned body tissue may be cultured over a period of time toallow changes in the biochemical composition and histoarchitecture tooccur. Preferably, the conditioned body tissue is allowed a period oftime to produce a biological material in an amount that is differentthan the amount that would be produce by an unconditioned body tissue.

The period of time in culture varies depending on the type ofconditioning and also the extent of change desired. In specificembodiments, the conditioned body tissue may be cultured for at least 10minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, atleast 12 hours, at least 24 hours, at least 2 days, at least 4 days, atleast 6 days, at least 8 days, at least 10 days, at least 1 week, atleast 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, orat least 3 months.

For conditioning process that are carried out in vitro, the body tissuemay be grown in multicavity bag or bioreactors which provide low shearto the tissue. The bioreactor designs useful for the present inventionare disclosed in, e.g., U.S. Pat. Nos. 4,988,623; 5,026,650; 5,153,131;and 5,928,945. In a preferred embodiment, a horizontal rotating wallvessel (RWV) bioreactor is used. The RWV bioreactor is described in U.S.Pat. No. 5,026,650 and is incorporated by reference herein. In preferredembodiments, culture medium such as supplemented Dulbeccos modifiedEagle medium (DMEM) (e.g., Life Technologies, Grand Island, N.Y.) may beused.

4.1.4 Assays for Monitoring the Effects of Conditioning

Changes in the amount of biologically active material subsequent tovarious conditioning methods may be assayed using methods known in theart. For example, mRNA levels for any factors may be determined usingstandard techniques in the art such as the quantitative reversetranscript TaqMan® polymerase chain reaction (QRTPCR) (see, e.g.,Holland et al., 1991, Proc. Natl. Acad. Sci. USA 88:7276-7289 and Lee etal., 1993, Nucl. Acids Res. 21:3761-3766). Protein levels can also bedetermined by techniques such as Western blots, standard ELISA assays,and biological activity assays such as the chick chorioallantoicmembrane (CAM) assay.

4.1.5 Decellularized Extracellular Matrix

Decellularization generally refers to the removal of all cells, cellularcomponents, and other non-extracellular matrix components (e.g., serum,fat) while leaving intact an extracellular matrix (ECM) component. It isbelieved that the process of decellularization can reduce or eliminateimmune response associated with the cells as well as the cellularcomponents. Acellular vascular tissues have been suggested to be idealnatural biomaterials for tissue repair and engineering (Schmidt andBaier, 2000, Biomaterials 21:2215-31).

Several means of reducing the viability of native cells in tissues andorgans are known, including physical, chemical, and biological methods(see, e.g. Kaushal et al., 2001, Nature Medicine 7(9):1035; Schmidt etal., supra; and U.S. Pat. No. 5,192,312, which are incorporated hereinby reference). Such methods may be employed in accordance with theprocess described herein. However, in preferred embodiments, thedecellularization technique employed should not result in grossdisruption of the anatomy of the body tissue or substantially alter itsbiomechanical properties or histoarchitecture. Similarly, the treatmentof the body tissue to produce a decellularized extracellular matrixshould also not leave a cytotoxic environment that inhibit subsequentrepopulation of the extracellular matrix with cells from a recipientafter implantation of the decellularized extracellular matrix.Decellularization by physical, chemical and/or biological treatments areoptimized to preserve as much as possible the biological material ofinterest and more importantly, the microstructure of the extracellularmatrix.

Extracellular matrix may be isolated from the conditioned body tissueusing a physical technique, including but not limited to centrifugation,rinsing, agitation, freeze-thaw, sedimentation, dialysis, electricalstimulation, electromagnetic forces, hydrostatic or hydrodynamic forces,blasting with sound waves, and ultrasonication. For example, theconditioned body tissue may be minced to disrupt the cell membrane anddisorganize cellular components. The minced body tissue may then becentrifuged with a liquid preparation, preferably Histopaque and morepreferably water or saline, which separates components of differentdensities. In preferred embodiments, the speed for centrifugation rangesfrom 100 to 10,000 g, and more preferably, from 2,500 to 7,500 g, forbetween 5 to 20 minutes. The components in the resulting suspension maythen be separated using filters of specific pore size. In oneembodiment, the filter is of a pore size, preferably of 70 to 250 μm,that allows the extracellular matrix to pass through. In anotherembodiment, the filter is of a pore size, preferably of 20 to 100 μm,that retains the extracellular matrix and larger components. Filtrationis carried out in one step or a series of steps.

It has been reported that modification of the magnitude of the membranedipole potential using compounds such as cholesterol, phloretin, and6-ketocholestanol may also influence binding capacity and disruptsmembrane domains. (Asawakarn T. et al., 2001, J Biol. Chem.276:38457-63). Accordingly, the present invention further relates tomethods for decellularizing conditioned body tissue by agitatingcellular membrane potential using electrical (e.g., voltage) means.

In another specific embodiment, the formation of intracellular ice isused to decellularize the conditioned body tissue. For example, vaporphase freezing (slow rate of temperature decline) of the body tissuereduces the cellularity of the body tissue as compared to liquid phasefreezing (rapid). However, slow freezing processes, in the absence ofcryoprotectant, may result in tissue disruption such as cracking.Colloid-forming materials may be added during freeze-thaw cycles toalter ice formation patterns in the body tissue. Polyvinylpyrrolidone(10% w/v) and dialyzed hydroxyethyl starch (10% w/v) may be added tostandard cryopreservation solutions (DMEM, 10% DMSO, 10% fetal bovineserum) to reduce extracellular ice formation while permitting formationof intracellular ice. This allows a measure of decellularization whileaffording the collagenase tissue matrix some protection from ice damage.

Alternatively, the conditioned body tissue may be decellularized using achemical technique. In one embodiment, the conditioned body tissue istreated with a solution effective to lyse native cells. Preferably, thesolution may be an aqueous hypotonic or low ionic strength solutionformulated to effectively lyse the native tissue cells. Such an aqueoushypotonic solution may be de-ionized water or an aqueous hypotonicbuffer. Preferably, the aqueous hypotonic buffer may contain additivesthat provide suboptimal conditions for the activity of selectedproteases, e.g., collagenase, which may be released as a result ofcellular lysis. Additives such as metal ion chelators, e.g.,1,10-phenanthroline and ethylenediaminetetraacetic acid (EDTA), createan environment unfavorable to many proteolytic enzymes.

In another embodiment, the conditioned body tissue is treated with ahypotonic lysis solution with protease inhibitors. General inhibitorsolutions manufactured by Sigma and Genotech are preferred.Specifically, 4-(2-aminoethyl)-benzene-sulfonyl fluoride, E-64,bestatin, leopeptin, aprotin, PMSF, Na EDTA, TIMPs, pepstatin A,phosphoramidon, and 1,10-phenanthroline are non-limiting examples ofpreferred protease inhibitors. The hypotonic lysis solution may haveinclude a buffered solution of water, pH 5.5 to 8, preferably pH 7 to 8.In preferred embodiments, the hypotonic lysis solution is free fromcalcium and zinc ions. Additionally, control of the temperature and timeparameters during the treatment of the body tissue with the hypotoniclysis solution, may also be employed to limit the activity of proteases.

In certain embodiments, the body tissue is treated with a detergent. Inone embodiment, the body tissue is treated with an anionic detergent,preferably sodium dodecyl sulfate in buffer. In another embodiment, thebody tissue is treated with a non-ionic detergent, such as Triton X-100or 1% octyl phenoxyl polyethoxyethanol, to solubilize cell membranes andfat. In a preferred embodiment, the body tissue is treated with acombination of different classes of detergents, for example, a nonionicdetergent, Triton X-100, and an anionic detergent, sodium dodecylsulfate, to disrupt cell membranes and aid in the removal of cellulardebris from tissue.

Steps should be taken to eliminate any residual detergent levels in theextracellular matrix, so as to avoid interference with the latter'sability to repair, regenerate or strengthen defective, diseased, damagedor ischemic tissues or organs. Selection of detergent type andconcentration will be based partly on its preservation of the structure,composition, and biological activity of the extracellular matrix.

In other embodiments, extracellular matrix may be isolated from theconditioned body tissue using a biological technique. Various enzymesmay be used to eliminate viable native cells from the body tissue.Preferably, the enzyme treatment limits the generation of newimmunological sites. For instance, extended exposure of the body tissueto proteases such as trypsin result in cell death. However, because atleast a portion of the type I collagen molecule is sensitive to avariety of proteases, including trypsin, this may not be the approach ofchoice for collagenous grafts intended for implant in high mechanicalstress locations.

In one embodiment, the body tissue is treated with nucleases to removeDNA and RNA. Nucleases are effective to inhibit cellular metabolism,protein production, and cell division without degrading the underlyingcollagen matrix. Nucleases that can be used for digestion of native cellDNA and RNA include both exonucleases and endonucleases. A wide varietyof which are suitable for use in this step of the process and arecommercially available. For example, exonucleases that effectivelyinhibit cellular activity include DNase I and RNase A (SIGMA ChemicalCompany, St. Louis, Mo.) and endonucleases that effectively inhibitcellular activity include EcoR I (SIGMA Chemical Company, St. Louis,Mo.) and Hind III (SIGMA Chemical Company, St. Louis, Mo.). It ispreferable that the selected nucleases are applied in a physiologicalbuffer solution which contains ions, such as magnesium and calciumsalts, which are optimal for the activity of the nuclease. It is alsopreferred that the ionic concentration of the buffered solution, thetreatment temperature, and the length of treatment are selected toassure the desired level of effective nuclease activity. The buffer ispreferably hypotonic to promote access of the nucleases to the cellinteriors.

Other enzymatic digestion may be suitable for use herein, for example,enzymes that disrupt the function of native cells in a transplant tissuemay be used. For example, phospholipase, particularly phospholipases Aor C, in a buffered solution, may be used to inhibit cellular functionby disrupting cellular membranes of endogenous cells. Preferably, theenzyme employed should not have a detrimental effect on theextracellular matrix protein. The enzymes suitable for use may also beselected with respect to inhibition of cellular integrity, and alsoinclude enzymes which may interfere with cellular protein production.The pH of the vehicle, as well as the composition of the vehicle, willalso be adjusted with respect to the pH activity profile of the enzymechosen for use. Moreover, the temperature applied during application ofthe enzyme to the tissue should be adjusted in order to optimizeenzymatic activity.

In another embodiment, the body tissue is treated so the cells areremoved using immunomagnetic bead separation techniques directed to cellsurface markers (e.g., integrins, lineage markers, stem cell markers).Immunomagnetic separation (IMS) technology can isolate strainspossessing specific and characteristic surface antigens (Olsvik O. etal., 1994, Clin. Microbiol Rev. 7:43-54). Commercially availableimmunomagnetic separation processes such as Cell Release™ (SigrisResearch, Brea, Calif.) was developed to address the need for a fast,general-purpose way to detach intact cells from beads afterimmunomagnetic separation.

Subsequent to decellularization protocols, the resultant extracellularmatrix is washed at least once with suitable chemical solutions, such assaline, protease, enzymes, detergents, alcohols, acidic or basicsolutions, salt solutions, etc., to assure removal of cell debris whichmay include cellular protein, cellular lipids, and cellular nucleicacid, as well as any extracellular debris such as lipids andproteoglycans. Removal of the cellular and extracellular debris reducesthe likelihood of the extracellular matrix eliciting an adverse immuneresponse from the recipient upon injection or implantation. For example,the tissue may be incubated in a balanced salt solution such as Hanks'Balanced Salt Solution (HBSS), preferably sterile. The washing processmay include incubation at a temperature of between about 2° C. and 42°C., with 4° C. to 25° C. most preferable. The transplant tissue matrixmay be incubated in the balanced salt wash solution for up to 10 to 12days, with changes in wash solution every second or third day. Thecomposition of the balanced salt solution wash, and the conditions underwhich it is applied to the transplant tissue matrix may be selected todiminish or eliminate the activity of the nuclease or other enzymeutilized during the decellularization process.

Optionally, an antibacterial, an antifungal or a sterilant or acombination thereof, may be included in the balanced salt wash solutionto protect the transplant tissue matrix from contamination withenvironmental pathogens. In certain embodiments, the ECM is sterilizedby irradiation, ultraviolet light exposure, ethanol incubation(70-100%), treatment with glutaraldehyde, peracetic acid (0.1-1% in 4%ethanol), chloroform (0.5%), or antimycotic and antibacterialsubstances.

The extracellular matrix prepared in accordance with the above is freeof its native cells, and additionally, cellular and extra-cellularantigen components have been washed out of the extracellular matrix.Preferably, the extracellular matrix has been treated in a manner whichlimits the generation of new immunological sites in the collagen matrix.The ECM is obtained as a slurry of small particles. This slurry mayeventually be processed into an implant.

In addition, the decellularized extracellular matrix may contain asignificant portion of the original tissue mass retaining physicalproperties in regard to strength and elasticity and has components whichare largely collagens but also comprise glycosaminoglycans and proteinsclosely associated with collagen such as the basement membrane complex,laminin and fibronectin.

One aspect of the invention further provides the preservation of thedecellularized extracellular matrix for later use. The decellularizedextracellular matrix can be freeze-dried for prolonged storage.Likewise, the decellularized extracellular matrix can be air-dried byany known standard techniques. In one embodiment, the decellularizedextracellular matrix can be concentrated or dehydrated and laterreconstituted or rehydrated, respectively, before use. In yet anotherembodiment, the decellularized extracellular matrix can be used toscreen pathogens such as bacteria, virus, and fungus, etc.

In yet another embodiment, the decellularized extracellular matrix islyophilized. The lyophilized ECM may be in the form of an implant whichhas pores. Characteristics of the pore structure can be controlled byprocess parameters. In yet another embodiment, the decellularizedextracellular matrix is formed as a gel. Preferably, the proteins aretemporarily and reversibly denatured. In yet another embodiment, thedecellularized extracellular matrix is precipitated or co-precipitatedwith other proteins or biologics.

In certain embodiments, the decellularized extracellular matrix iscryopreserved. General techniques for cryopreservation of cells arewell-known in the art (see, e.g., Doyle et al., (eds), 1995, Cell &Tissue Culture: Laboratory Procedures, John Wiley & Sons, Chichester;and Ho and Wang (eds), 1991, Animal Cell Bioreactors,Butterworth-Heinemann, Boston, each of which is incorporated herein byreference). Preferably, the tissue or organ is thawed rapidly beforeuse, in a water bath at 34° C. to 37° C., to avoid damage to the cells.Cryopreservation of decellularized extracellular matrix would assure asupply or inventory of substantially non-immunogenic extracellularmatrices which, upon thawing, would be ready for further treatmentaccording to the subsequent steps of this invention, or furtherprocessed as desired to provide an implant tissue product. For example,extracellular matrices may be inventoried until such time as theparticular cells to be employed during repopulation are identified. Thismay be of particular utility when the extracellular matrix is to berepopulated with cells derived from the recipient or other cellsselected for use based on their immunological compatibility with aspecific recipient. The ECM may also be used in combination with cells.

4.2 Uses of the Decellularized Extracellular Matrix

The present invention further provides methods for repairing,regenerating or strengthening cells, tissues or organs. In particular,the invention relates to methods for formulating the decellularizedextracellular matrix as pharmaceutical compositions, body implants,tissue regeneration scaffolds, and medical devices.

In certain embodiments, the decellularized extracellular matrix ofconditioned body tissue may be used to treat defective, diseased,damaged or ischemic tissues or organs which include, but are not limitedto, head, neck, eye, mouth, throat, esophagus, chest, bone, ligament,cartilage, tendons, lung, colon, rectum, stomach, prostate, breast,ovaries, fallopian tubes, uterus, cervix, testicles or otherreproductive organs, hair follicles, skin, diaphragm, thyroid, blood,muscles, bone marrow, heart, lymph nodes, blood vessels, largeintestine, small intestine, kidney, liver, pancreas, brain, spinal cord,and the central nervous system.

In particular, the decellularized extracellular matrix of conditionedbody tissue of the present invention may be used to treat diseases thatmay benefit from improved angiogenesis, cell proliferation and tissueregeneration. Such diseases or conditions include, but are not limitedto, burns, ulcer, trauma, wound, bond fracture, diabetes, psoriasis,arthritis, asthma, cystitis, inflammation, infection, ischemia,restenosis, stricture, atherosclerosis, occlusion, stroke, infarct,aneurysm, abdominal aortic aneurysm, uterine fibroid, urinaryincontinence, vascular disorders, hemophilia, cancer, and organ failure(e.g., heart, kidney, lung, liver, intestine, etc.).

In a specific embodiment, the present invention regenerates or replacesat least 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, at least 10%, at least 5%, or at least 1% of defective,diseased, damaged or ischemic cells from the affected tissue or organ.

The methods of the present invention is provided for an animal,including but not limited to mammals such as a non-primate (e.g., cows,pigs, horses, chickens, cats, dogs, rats, etc.), and a primate (e.g.monkey such as acynomolgous monkey and a human). In a preferredembodiment, the subject is a human.

The present invention is useful alone or in combination with othertreatment modalities. In certain embodiments, the treatment of thepresent invention further includes the administration of one or moreimmunotherapeutic agents, such as antibodies and immunomodulators, whichinclude, but are not limited to, HERCEPTIN®, RITUXAN®, OVAREX™,PANOREX®, BEC2, IMC-C225, VITAXIN™, CAMPATH® I/H, Smart MI95,LYMPHOCIDE™, Smart I D10, ONCOLYM™, rituximab, gemtuzumab, ortrastuzumab. In certain other embodiments, the treatment method furthercomprises hormonal treatment. Hormonal therapeutic treatments comprisehormonal agonists, hormonal antagonists (e.g., flutamide, tamoxifen,leuprolide acetate (LUPRON™), LH-RH antagonists), inhibitors of hormonebiosynthesis and processing, steroids (e.g., dexamethasone, retinoids,betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone,glucocorticoids, mineralocorticoids, estrogen, testosterone,progestins), antigestagens (e.g., mifepristone, onapristone), andantiandrogens (e.g., cyproterone acetate).

4.2.1 Pharmaceutical Compositions

The decellularized extracellular matrix of conditioned body tissue canbe formulated into pharmaceutical compositions that are suitable foradministration to a subject. Such compositions comprise aprophylactically or therapeutically effective amount of thedecellularized extracellular matrix as disclosed herein, and apharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete) or, more preferably, MF59C.1 adjuvantavailable from Chiron, Emeryville, Calif.), excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Other examples of suitable pharmaceutical vehicles aredescribed in “Remington: the Science and Practice of Pharmacy”, 20thed., by Mack Publishing Co. 2000.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed from an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

Various delivery systems are known and can be used to administer thecompositions of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis (see, e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), etc. Methods ofadministering a prophylactic or therapeutic amount of the compositionsof the invention include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intracoronary,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal, inhaled, and oral routes). The composition comprisingdecellularized extracellular matrix of conditioned body tissue may beadministered by any convenient route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents, preferablypaclitaxel. Administration can be systemic or local. In addition, it maybe desirable to introduce the pharmaceutical composition of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

In another embodiment, the decellularized extracellular matrix ofconditioned body tissue can be delivered in a controlled release orsustained release system. In one embodiment, a pump may be used toachieve controlled or sustained release (see Langer, 1990, Science249:1527-1533; Sefton, 1987, CRC Crit. Ref: Biomed. Eng. 14:20; Buchwaldet al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.321:574). Any technique known to one of skill in the art can be used toproduce sustained release formulations comprising the decellularizedextracellular matrix of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

In another embodiment, polymeric materials can be used to achievecontrolled or sustained release of the decellularized extracellularmatrix material (see, e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, N.Y. (1984); Ranger and Peppas, 1983, J. Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015;5,989,463; and 5,128,326; International Publication Nos. WO 99/15154 andWO 99/20253). Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glucosides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable during storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity to the target, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, 1984, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138).

The amount of the pharmaceutical composition which will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro assays and animal models mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

4.2.2 Body Implant

Methods of the present invention also include methods for making andimplanting a body implant comprising the decellularized extracellularmatrix of conditioned body tissue.

The body implants of the present invention may be, without limitation:(1) vascular implants, such as carotid artery replacement, and generalvein and artery replacement in the body; (2) heart valves and patches;(3) burn dressings and coverings; (4) muscle, tooth and bone implants;(5) pericardium and membranes; (6) myocardial patch; (7) urethral sling;and (8) fiber for filling aneurysms.

The modified body implants comprising decellularized extracellularmatrix can be implanted in vivo at the site of tissue damage promoterepair, regeneration and/or strengthening. In addition, the materialsand methods of this invention are useful to promote the in vitro cultureand differentiation of cells and tissues.

4.2.3 Tissue Regeneration Scaffold

One aspect of the invention provides for the incorporation of thedecellularized extracellular matrix of conditioned body tissue into abiocompatible material for implantation into a subject, preferablyhuman. In a preferred embodiment, the biocompatible material is in theform of a scaffold.

The scaffold may be of natural collagen, decellularized, conditionedextracellular matrix, or synthetic polymer. In certain preferredembodiments, the scaffold serves as a template for cell proliferationand ultimately tissue formation. In a specific embodiment, the scaffoldallows the slow release of the decellularized extracellular matrix ofthe invention into the surrounding tissue. As the cells in thesurrounding tissue begin to multiply, they fill up the scaffold and growinto three-dimensional tissue. Blood vessels then attach themselves tothe newly grown tissue, the scaffold dissolves, and the newly growntissue eventually blends in with its surrounding.

4.2.4 Medical Device Comprising Decellularized Extracellular Matrix

The decellularized extracellular matrix of the invention may be used toform a medical or prosthetic device, preferably a stent or an artificialheart, which may be implanted in the subject. More specifically, thedecellularized extracellular matrix of the invention may be incorporatedinto the base material needed to make the device. For example, in stentcomprising a sidewall of elongated members or wire-like elements, thedecellularized extracellular matrix material can be used to form theelongated members or wire-like elements. On the other hand, thedecellularized ECM material of the invention can be used to coat orcover the medical device.

The medical devices of the present invention may be inserted orimplanted into the body of a patient.

4.2.4.1 Types of Medical Device

Medical devices that are useful in the present invention can be made ofany biocompatible material suitable for medical devices in general whichinclude without limitation natural polymers, synthetic polymers,ceramics and metallics. Metallic material is more preferable. Suitablemetallic materials include metals and alloys based on titanium (such asnitinol, nickel titanium alloys, thermo-memory alloy materials),stainless steel, tantalum, nickel-chrome, or certain cobalt alloysincluding cobalt-chromium-nickel alloys such as Elgiloy® and Phynox®.Metallic materials also include clad composite filaments, such as thosedisclosed in WO 94/16646.

Metallic materials may be made into elongated members or wire-likeelements and then woven to form a network of metal mesh. Polymerfilaments may also be used together with the metallic elongated membersor wire-like elements to form a network mesh. If the network is made ofmetal, the intersection may be welded, twisted, bent, glued, tied (withsuture), heat sealed to one another; or connected in any manner known inthe art.

The polymer(s) useful for forming the medical device should be ones thatare biocompatible and avoid irritation to body tissue. They can beeither biostable or bioabsorbable. Suitable polymeric materials includewithout limitation polyurethane and its copolymers, silicone and itscopolymers, ethylene vinyl-acetate, polyethylene terephtalate,thermoplastic elastomers, polyvinyl chloride, polyolefins, cellulosics,polyamides, polyesters, polysulfones, polytetrafluorethylenes,polycarbonates, acrylonitrile butadiene styrene copolymers, acrylics,polylactic acid, polyglycolic acid, polycaprolactone, polylacticacid-polyethylene oxide copolymers, cellulose, collagens, and chitins.

Other polymers that are useful as materials for medical devices includewithout limitation dacron polyester, poly(ethylene terephthalate),polycarbonate, polymethylmethacrylate, polypropylene, polyalkyleneoxalates, polyvinylchloride, polyurethanes, polysiloxanes, nylons,poly(dimethyl siloxane), polycyanoacrylates, polyphosphazenes,poly(amino acids), ethylene glycol I dimethacrylate, poly(methylmethacrylate), poly(2-hydroxyethyl methacrylate),polytetrafluoroethylene poly(HEMA), polyhydroxyalkanoates,polytetrafluorethylene, polycarbonate, poly(glycolide-lactide)co-polymer, polylactic acid, poly(ε-caprolactone),poly(β-hydroxybutyrate), polydioxanone, poly(γ-ethyl glutamate),polyiminocarbonates, poly(ortho ester), polyanhydrides, alginate,dextran, chitin, cotton, polyglycolic acid, polyurethane, or derivatizedversions thereof, i.e., polymers which have been modified to include,for example, attachment sites or cross-linking groups, e.g., RGD, inwhich the polymers retain their structural integrity while allowing forattachment of molecules, such as proteins, nucleic acids, and the like.

Furthermore, although the invention can be practiced by using a singletype of polymer to form the medical device, various combinations ofpolymers can be employed. The appropriate mixture of polymers can becoordinated to produce desired effects when incorporated into a medicaldevice. In certain preferred embodiments, the decellularizedextracellular matrix is mixed with a polymer.

The decellularized extracellular matrix of the invention may also beused alone or in combination with a polymer described above to form themedical device. The decellularized extracellular matrix may be dried toincrease its mechanical strength. The dried decellularized extracellularmatrix may then be used as the base material to form a whole or part ofthe medical device. In preferred embodiments, the decellularizedextracellular matrix constitutes at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90%, at least 95%, at least99% by weight or by size of the medical device.

Examples of the medical devices suitable for the present inventioninclude, but are not limited to, stents, surgical staples, catheters(e.g., central venous catheters and arterial catheters), guidewires,cannulas, cardiac pacemaker leads or lead tips, cardiac defibrillatorleads or lead tips, implantable vascular access ports, blood storagebags, blood tubing, vascular or other grafts, intra-aortic balloonpumps, heart valves, cardiovascular sutures, total artificial hearts andventricular assist pumps, and extra-corporeal devices such as bloodoxygenators, blood filters, hemodialysis units, hemoperfusion units andplasmapheresis units.

Medical devices of the present invention include those that have atubular or cylindrical-like portion. The tubular portion of the medicaldevice need not to be completely cylindrical. For instance, thecross-section of the tubular portion can be any shape, such asrectangle, a triangle, etc., not just a circle. Such devices include,without limitation, stents and grafts. A bifurcated stent is alsoincluded among the medical devices which can be fabricated by the methodof the present invention.

Medical devices which are particularly suitable for the presentinvention include any kind of stent for medical purposes which is knownto the skilled artisan. Suitable stents include, for example, vascularstents such as self-expanding stents and balloon expandable stents.Examples of self-expanding stents useful in the present invention areillustrated in U.S. Pat. Nos. 4,655,771 and 4,954,126 issued to Wallstenand 5,061,275 issued to Wallsten et al. Examples of appropriateballoon-expandable stents are shown in U.S. Pat. No. 5,449,373 issued toPinchasik et al.

4.2.4.2 Methods of Coating the Medical Device

In the present invention, the decellularized extracellular matrix of theinvention, preferably in combination with a biologically active materialsuch as paclitaxel, can be applied by any method to a surface of amedical device to form a coating. Examples of suitable methods arespraying, laminating, pressing, brushing, swabbing, dipping, rolling,electrostatic deposition and all modern chemical ways of immobilizationof bio-molecules to surfaces. Preferably, the decellularizedextracellular matrix is applied to a surface of a medical device byspraying, rolling, laminating, and pressing. In one embodiment of thepresent invention, more than one coating method can be used to make amedical device. In certain embodiments, the decellularized extracellularmatrix is placed into a carrier in order to apply it to the devicesurface. Non-limiting examples of carriers include SIBS, PLGA, PGA,collagen (all types), etc.

Furthermore, before applying the coating composition, the surface of themedical device is optionally subjected to a pre-treatment, such asroughening, oxidizing, sputtering, plasma-deposition or priming inembodiments where the surface to be coated does not comprisedepressions. Sputtering is a deposition of atoms on the surface byremoving the atom from the cathode by positive ion bombardment through agas discharge. Also, exposing the surface of the device to a primer is apossible method of pre-treatment.

Coating compositions suitable for applying coating materials to thedevices of the present invention can include a polymeric material andpreferably a biologically active material dispersed or dissolved in asolvent suitable for the medical device, which are known to the skilledartisan. The solvents used to prepare coating compositions include oneswhich can dissolve the polymeric material into solution or suspend thepolymeric material and do not alter or adversely impact the therapeuticproperties of the biologically active material employed. For example,useful solvents for silicone include tetrahydrofuran (THF), chloroform,toluene, acetone, isooctane, 1,1,1-trichloroethane, dichloromethane, andmixture thereof.

The polymeric material should be a material that is biocompatible andavoids irritation to body tissue. Preferably the polymeric materialsused in the coating composition of the present invention are selectedfrom the following: polyurethanes, silicones (e.g., polysiloxanes andsubstituted polysiloxanes), and polyesters. Also preferable as apolymeric material is styrene-isobutylene-styrene (SIBS). Other polymerswhich can be used include ones that can be dissolved and cured orpolymerized on the medical device or polymers having relatively lowmelting points that can be blended with biologically active materials.Additional suitable polymers include, thermoplastic elastomers ingeneral, polyolefins, polyisobutylene, ethylene-alphaolefin copolymers,acrylic polymers and copolymers, vinyl halide polymers and copolymerssuch as polyvinyl chloride, polyvinyl ethers such as polyvinyl methylether, polyvinylidene halides such as polyvinylidene fluoride andpolyvinylidene chloride, polyacrylonitrile, polyvinyl ketones, polyvinylaromatics such as polystyrene, polyvinyl esters such as polyvinylacetate, copolymers of vinyl monomers, copolymers of vinyl monomers andolefins such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS (acrylonitrile-butadiene-styrene)resins, ethylene-vinyl acetate copolymers, polyamides such as Nylon 66and polycaprolactone, alkyd resins, polycarbonates, polyoxymethylenes,polyimides, polyethers, epoxy resins, rayon-triacetate, cellulose,cellulose acetate, cellulose butyrate, cellulose acetate butyrate,cellophane, cellulose nitrate, cellulose propionate, cellulose ethers,carboxymethyl cellulose, collagens, chitins, polylactic acid,polyglycolic acid, polylactic acid-polyethylene oxide copolymers, EPDM(etylene-propylene-diene) rubbers, fluorosilicones, polyethylene glycol,polysaccharides, phospholipids, and combinations of the foregoing.

More preferably for medical devices which undergo mechanical challenges,e.g. expansion and contraction, the polymeric materials should beselected from elastomeric polymers such as silicones (e.g. polysiloxanesand substituted polysiloxanes), polyurethanes, thermoplastic elastomers,ethylene vinyl acetate copolymers, polyolefin elastomers, and EPDMrubbers. Because of the elastic nature of these polymers, the coatingcomposition is capable of undergoing deformation under the yield pointwhen the device is subjected to forces, stress or mechanical challenge.

The term “biologically active material” encompasses therapeutic agents,such as drugs, and also genetic materials and biological materials. Thegenetic materials mean DNA or RNA, including, without limitation, ofDNA/RNA encoding a useful protein stated below, intended to be insertedinto a human body including viral vectors and non-viral vectors. Thebiological materials include cells, yeasts, bacteria, proteins,peptides, cytokines and hormones. Examples for peptides and proteinsinclude vascular endothelial growth factor (VEGF), transforming growthfactor (TGF), fibroblast growth factor (FGF), epidermal growth factor(EGF), cartilage growth factor (CGF), nerve growth factor (NGF),keratinocyte growth factor (KGF), skeletal growth factor (SGF),osteoblast-derived growth factor (BDGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), cytokine growth factors (CGF),platelet-derived growth factor (PDGF), hypoxia inducible factor-1(HIF-1), stem cell derived factor (SDF), stem cell factor (SCF),endothelial cell growth supplement (ECGS), granulocyte macrophage colonystimulating factor (GM-CSF), growth differentiation factor (GDF),integrin modulating factor (IMF), calmodulin (CaM), thymidine kinase(TK), tumor necrosis factor (TNF), growth hormone (GH), bone morphogenicprotein (BMP) (e.g., BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7(PO-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-14, BMP-15, BMP-16,etc.), matrix metalloproteinase (MMP), tissue inhibitor of matrixmetalloproteinase (TIMP), cytokines, interleukin (e.g., IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15,etc.), lymphokines, interferon, integrin, collagen (all types), elastin,fibrillins, fibronectin, vitronectin, laminin, glycosaminoglycans,proteoglycans, transferrin, cytotactin, cell binding domains (e.g.,RGD), and tenascin. Currently preferred BMP's are BMP-2, BMP-3, BMP-4,BMP-5, BMP-6, BMP-7. These dimeric proteins can be provided ashomodimers, heterodimers, or combinations thereof, alone or togetherwith other molecules. Cells can be of human origin (autologous orallogeneic) or from an animal source (xenogeneic), geneticallyengineered, if desired, to deliver proteins of interest at thetransplant site. The delivery media can be formulated as needed tomaintain cell function and viability. Cells include progenitor cells(e.g., endothelial progenitor cells), stem cells (e.g., mesenchymal,hematopoietic, neuronal), stromal cells, parenchymal cells,undifferentiated cells, fibroblasts, macrophage, and satellite cells.Biologically active materials also include non-genetic therapeuticagents, such as:

-   -   anti-thrombogenic agents such as heparin, heparin derivatives,        urokinase, and PPack (dextrophenylalanine proline arginine        chloromethylketone);    -   anti-proliferative agents such as enoxaprin, angiopeptin, or        monoclonal antibodies capable of blocking smooth muscle cell        proliferation, hirudin, and acetylsalicylic acid, amlodipine and        doxazosin;    -   anti-inflammatory agents such as glucocorticoids, betamethasone,        dexamethasone, prednisolone, corticosterone, budesonide,        estrogen, sulfasalazine, and mesalamine;    -   antineoplastic/antiproliferative/anti-mitotic agents such as        paclitaxel, 5-fluorouracil, cisplatin, vinblastine, cladribine,        vincristine, epothilones, methotrexate, azathioprine, adriamycin        and mutamycin; endostatin, angiostatin and thymidine kinase        inhibitors, taxol and its analogs or derivatives;    -   anesthetic agents such as lidocaine, bupivacaine, and        ropivacaine;    -   anti-coagulants such as D-Phe-Pro-Arg chloromethyl keton, an RGD        peptide-containing compound, heparin, antithrombin compounds,        platelet receptor antagonists, anti-thrombin antibodies,        anti-platelet receptor antibodies, aspirin (aspirin is also        classified as an analgesic, antipyretic and anti-inflammatory        drug), dipyridamole, protamine, hirudin, prostaglandin        inhibitors, platelet inhibitors and tick antiplatelet peptides;    -   DNA demethylating drugs such as 5-azacytidine, which is also        categorized as a RNA or DNA metabolite that inhibit cell growth        and induce apoptosis in certain cancer cells;    -   vascular cell growth promoters such as growth factors, vascular        endothelial growth factors (VEGF, all types including VEGF-2),        growth factor receptors, transcriptional activators, and        translational promoters;    -   vascular cell growth inhibitors such as antiproliferative        agents, growth factor inhibitors, growth factor receptor        antagonists, transcriptional repressors, translational        repressors, replication inhibitors, inhibitory antibodies,        antibodies directed against growth factors, bifunctional        molecules consisting of a growth factor and a cytotoxin,        bifunctional molecules consisting of an antibody and a        cytotoxin;    -   cholesterol-lowering agents; vasodilating agents; and agents        which interfere with endogenous vasoactive mechanisms;    -   anti-oxidants, such as probucol;    -   antibiotic agents, such as penicillin, cefoxitin, oxacillin,        tobranycin, rapamycin;    -   angiogenic substances, such as acidic and basic fibroblast        growth factors, estrogen including estradiol (E2), estriol (E3)        and 17-Beta Estradiol;    -   drugs for heart failure, such as digoxin, beta-blockers,        angiotensin-converting enzyme (ACE) inhibitors including        captopril, enalopril, and statins and related compounds; and

In certain embodiments, the medical device of the present invention iscovered with one coating layer. In certain other embodiments, themedical device of the present invention is covered with more than onecoating layer. In preferred embodiments, the medical device is coveredwith different coating layers. For example, the coating can comprise afirst layer and a second layer that contain different biologicallyactive materials. Alternatively, the first layer and the second layermay contain an identical biologically active material having differentconcentrations. In one embodiment, either the first layer or the secondlayer may be free of biologically active material.

5. EXAMPLES

5.1 Increased VEGF Levels in Submucosa

Porcine intestines are conditioned with plasmid DNA encoding human VEGF.The DNA is delivered using a drug delivery balloon (Remedy, BostonScientific, Natick, Mass.) which is placed in the intestine. Followinginfusion, the animal is allowed to live normally for one week. Afterthis time, the animal is sacrificed and the targeted region of theintestine is isolated. Finally, the submucosal layer is isolated fromthe muscular layers and further processed to remove cells. Thetransfection with DNA results in higher levels of VEGF in the tissue,and hence an improved tissue regeneration scaffold. Ultrasound oriontophoresis may be used to improve conditioning of the tissue. Thesetechniques are used during delivery of the DNA to enhance diffusion intothe tissue and potentially increase transfection by disrupting cellmembranes.

6. EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings using no more thanroutine experimentation. Such modifications and equivalents are intendedto fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1. A method for producing a decellularized extracellular matrix materialcontaining a biological material, wherein the method comprises: (a)conditioning body tissue of a donor animal to produce the biologicalmaterial in an amount different than the amount of the biologicalmaterial that the body tissue would produce absent the conditioning; (b)allowing the conditioned body tissue to produce the biological material;(c) harvesting the conditioned body tissue from the donor animal; and(d) decellularizing the conditioned body tissue to obtain theextracellular matrix material containing the biological material.
 2. Themethod of claim 1, wherein steps (a) and (b) are conducted before theharvesting in step (c).
 3. The method of claim 1, wherein steps (a) and(b) are conducted after the harvesting in step (c).
 4. The method ofclaim 3, wherein step (b) comprises culturing the conditioned bodytissue in a bioreactor to allow the conditioned body tissue to producethe biological material.
 5. The method of claim 1 further comprisingmonitoring the amount of biological material produced by the conditionedbody tissue.
 6. The method of claim 1 further comprising delivering atherapeutic agent to the body tissue before the conditioning in step(a).
 7. The method of claim 1 further comprising delivering atherapeutic agent to the body tissue after the conditioning in step (a).8. The method of claim 1 further comprising adding a therapeutic agentto the decellularized extracellular matrix material.
 9. The method ofclaim 1, wherein the donor animal is a mammal.
 10. The method of claim1, wherein the mammal is selected from the group consisting of cows,pigs, horses, chickens, cats, dogs, rats, monkeys, and humans.
 11. Themethod of claim 1, wherein the body tissue is selected from the groupconsisting of epithelial tissue, connective tissue, muscle tissue, andnerve tissue.
 12. The method of claim 1, wherein the body tissue isselected from the group consisting of lymph vessels, blood vessels,heart valves, myocardium, pericardium, pericardial sac, dura mater,meniscus, omentum, mesentery, conjunctiva, umbilical cords, bone marrow,bone pieces, ligaments, tendon, tooth implants, dermis, skin, muscle,nerves, spinal cord, pancreas, gut, intestines, peritoneum, submucosa,stomach, liver, and bladder.
 13. The method of claim 1, wherein thebiological material is selected from the group consisting of vascularendothelial growth factor (VEGF), transforming growth factor (TGF),fibroblast growth factor (FGF), epidermal growth factor (EGF), cartilagegrowth factor (CGF), nerve growth factor (NGF), keratinocyte growthfactor (KGF), skeletal growth factor (SGF), osteoblast-derived growthfactor (BDGF), hepatocyte growth factor (HGF), insulin-like growthfactor (IGF), cytokine growth factors (CGF), platelet-derived growthfactor (PDGF), hypoxia inducible factor-1 (HIF-1), stem cell derivedfactor (SDF), stem cell factor (SCF), endothelial cell growth supplement(ECGS), granulocyte macrophage colony stimulating factor (GM-CSF),growth differentiation factor (GDF), integrin modulating factor (IMF),calmodulin (CaM), thymidine kinase (TK), tumor necrosis factor (TNF),growth hormone (GH), bone morphogenic proteins (BMP), matrixmetalloproteinase (MMP), tissue inhibitor of matrix metalloproteinase(TIMP), interferon, interleukins, cytokines, integrin, collagen (alltypes), elastin, fibrillins, fibronectin, laminin, glycosaminoglycans,vitronectin, proteoglycans, transferrin, cytotactin, cell bindingdomains (e.g., RGD), tenascin, and lymphokines.
 14. The method of claim1, wherein the body tissue is conditioned by a process selected from thegroup consisting of biological conditioning, chemical conditioning,pharmaceutical conditioning, physiological conditioning, and mechanicalconditioning.
 15. The method of claim 14, wherein the biologicalconditioning comprises transfecting the body tissue with a nucleic acidthat encodes the biological material.
 16. The method of claim 14,wherein the chemical conditioning comprises incubating the body tissuein a hypotonic or hypertonic solution.
 17. The method of claim 14,wherein the pharmaceutical conditioning comprises delivering atherapeutic agent to the body tissue.
 18. The method of claim 14,wherein the physiological conditioning comprises exposing the bodytissue to heat shock or cryopreservation followed by thawing.
 19. Themethod of claim 14, wherein the mechanical conditioning comprisesapplying a force to the body tissue.
 20. The method of claim 19, whereinthe force is selected from the group consisting of a mechanical force,centrifugal force, electrical force, electromagnetic force, hydrostaticor hydrodynamic force, sound wave, and ultrasound wave.
 21. Adecellularized extracellular matrix material produced by the method ofclaim 1 for injection into a subject.
 22. A decellularized extracellularmatrix material produced by the method of claim 1 for implantation intoa subject.
 23. A tissue regeneration scaffold for implantation into apatient comprising the decellularized extracellular matrix materialproduced by the method of claim
 1. 24. A method of using thedecellularized extracellular matrix material produced by the method ofclaim 1 to repair injured body tissue of a patient.
 25. A method ofusing the decellularized extracellular matrix material produced by themethod of claim 1 to regenerate injured body tissue of a patient.
 26. Amethod of using the decellularized extracellular matrix materialproduced by the method of claim 1 to strengthen injured body tissue of apatient.
 27. A method for producing a decellularized extracellularmatrix material containing a biological material, wherein the methodcomprises: (a) conditioning body tissue of a donor animal to produce thebiological material in an amount different than the amount of thebiological material that the body tissue would produce absent theconditioning, wherein the conditioning comprises transfecting the bodytissue with a nucleic acid that encodes the biological material; (b)allowing the conditioned body tissue to produce the biological material;(c) harvesting the conditioned body tissue from the donor animal; and(d) decellularizing the conditioned body tissue to obtain theextracellular matrix material containing the biological material. 28.The method of claim 27, wherein steps (a) and (b) are conducted beforethe harvesting in step (c).
 29. The method of claim 27, wherein steps(a) and (b) are conducted after the harvesting in step (c).
 30. Themethod of claim 27, wherein the biological material is vascularendothelial growth factor (VEGF).
 31. A method for producing adecellularized extracellular matrix material containing a biologicalmaterial, wherein the method comprises: (a) conditioning a body tissueof a donor animal to produce the biological material in an amountdifferent than the amount of the biological material that the bodytissue would produce absent the conditioning, wherein the conditioningcomprises applying a mechanical force to the body tissue; (b) allowingthe conditioned body tissue to produce the biological material; (c)harvesting the conditioned body tissue from the donor animal; and (d)decellularizing the conditioned body tissue to obtain the extracellularmatrix material containing the biological material.
 32. The method ofclaim 31, wherein steps (a) and (b) are conducted before the harvestingin step (c).
 33. The method of claim 31, wherein steps (a) and (b) areconducted after the harvesting in step (c).
 34. The method of 31,wherein the body tissue is small intestine tissue and the mechanicalforce is produced by the expansion of a balloon against the smallintestine tissue.
 35. A method for producing a tissue regenerationscaffold for implantation into a patient comprising: (a) conditioningbody tissue of a donor animal to produce the biological material in anamount different than the amount of the biological material that thebody tissue would produce absent the conditioning; (b) allowing theconditioned body tissue to produce the biological material; (c)harvesting the conditioned body tissue from the donor animal; (d)decellularizing the conditioned body tissue to obtain the extracellularmatrix material containing the biological material; and (e) forming thetissue regeneration scaffold from the decellularized extracellularmatrix material containing the biological material.
 36. A method forproducing a tissue regeneration scaffold for implantation into a patientcomprising: (a) harvesting body tissue from a donor animal; (b)conditioning the harvested body tissue in vitro to produce a biologicalmaterial in an amount different than the amount of the biologicalmaterial that the body tissue would produce absent the conditioning; (c)culturing the harvested and conditioned body tissue in a bioreactor toallow the body tissue to produce the biological material; (d)decellularizing the conditioned body tissue to obtain the extracellularmatrix material containing the biological material; and (e) forming thetissue regeneration scaffold from the decellularized extracellularmatrix material containing the biological material.
 37. An implantablemedical device comprising a surface and a decellularized extracellularmatrix material comprising a biological material, wherein thedecellularized matrix material is produced by a method comprising: (a)conditioning body tissue of a donor animal to produce the biologicalmaterial in an amount different than the amount of the biologicalmaterial that the body tissue would produce absent the conditioning; (b)allowing the conditioned body tissue to produce the biological material;(c) harvesting the body tissue from the donor animal; and (d)decellularizing the conditioned body tissue to obtain the extracellularmatrix material containing the biological material.
 38. The device ofclaim 37, wherein the decellularized extracellular matrix material isdisposed upon the surface of the device.
 39. The device of claim 37,wherein the device is a stent.
 40. The device of claim 37, wherein thedevice is an artificial heart.
 41. The device of claim 37, wherein thebiological material is elastin.